Animal feed composition for prevention of mycotoxicoses and method for preparing same



States "t t Patented Sept. 28, 1985 ANIMAL FEED COMPOSITION FORPREVENTKQN OF MYCOTOXICOSES AND METHOD EUR li -53E PARING SAME EoseplrForgaes, Pearl River, N.Y., assignor to American Qyanamid Company,fitanrford, Conrn, a corporation of Maine No Drawing. Filed Jan. 23,1963, Ser. No. 253,283

4 Claims. (Cl. 99-4) t This application is a continuation-in-part of mycopending application Serial No. 850,075, filed November 2, 1959, nowabandoned, which is a continuation-inpart of my application Serial No.582,323, filed May 3, 1956, now abandoned.

This invention relates to cereal compositions wherein the elaboration offungal metabolic by-products, which are toxic to animals ingesting thesame, is inhibited. Furthermore, the invention is adapted to animal feedcompositions containing 2-bromo-5-nitrothiazole for the prevention ofmycotoxicoses in domestic livestock and other animals.

Poultry hemorrhagic syndrome is an example of a mycotoxicoses and ischaracterized by certain abnormal clinical symptoms and by hemorrhagesin the legs, thighs and other organs, as well as by degenerative changesin the liver and kidneys, and by a pronounced depression v of theblood-forming tissues.

Although death losses as a result of the hemorrhagic syndrome range fromto over 50 percent of the afflicted birds and morbidity up to 90percent, the significance of this disease has even greater economicimplications. Many chickens which may appear clinically normal aredowngraded during dressing, because of hemorrhages in the musculature ofthe legs, thighs and breast.

In the past, various theories have been advanced as possible causes ofthis elusive disease, but with no conclusive evidence that any of theadvanced theoretical factors has been responsible for the classicalfield syndrome. For example, one of the most popular theories centers onavitaminosis K induced by sulfaquinoxaline toxicity, or by antagonism ofthis coccidiostat or other drugs to vitamin K. However, the fact thatthe syndrome has occurred in the complete absence of sulfaquinoxaline orother drugs regarded as antimetabolitic to vitamin K precludes thistheory. The avitaminosis K theory is invalidated further by the factthat very little beneficial response was elicited from theadministration of therapeutic quantities of vitamin K to the afflictedbirds. Therefore, methods used in the past for the amelioration of thishemorrhagic disease have not been successful.

I have discovered that toxic elaborated products as the result of growthof fungi on cereals fed to animals are the basic cause of thishemorrhagic disease. Thus, the hemorrhagic syndrome may be classified asbeing a mycotoxicoses, which is a toxemia produced in animals that haveconsumed toxic substances secreted by fungi. The relationship of thetoxic fungal metabolic products to the hemorrhagic syndrome has beenconfirmed conclusively by production of the hemorrhagic syndrome underlaboratory and field conditions.

Therefore, a principal object of my invention is the prevention of thosemaladies associated with ingestion of cereals, hereinafter defined, suchas straw, hay, grain, and processed feed on which fungi proliferate assaprophytes and give rise to toxic products, either by secretion ofmetabolic products into the substrata, or by the toxic substance beingpresent as an endotoxin in the mycelium or in the fruiting bodies of thefungus. Because of the possibility that small portions of infectedcereals may stand for a considerable period of time before theyingested, it is necessary that the cereals be protected againstdevelopment of toxic fungal metabolic by-products for a long period oftime under adverse conditions. It is with the prevention of such cerealborne intoxication that the present invention is particularly concerned.As used in this specification, the term cereal shall mean any grassyielding grain such as corn, wheat, or rice, also the grains soproduced; a prepared foodstuff of grain such as oatmeal or bran; theconversion product such as bread, as well as compositions thereof, andto include fabaceous plants and their products such as peanuts,soybeans, peanut meal and soybean meal and others.

Once having determined the etiology of the hemorrhagic syndrome andhaving classified it as a mycotoxicoses, I then discovered that theproduction of toxic materials, or at least toxic levels of thesematerials, could be suppressed by the addition ofZ-bromo-S-nitrothiazole to the cereal substrate. 1 further discoveredthat this could be accomplished by the addition of 10 to 500 ppm. of theZ-bromo-S-nitrothiazole to the cereal substrate. The preferredconcentration being within the range of 30 to 300 ppm.

It has been found that the occurrence of mycotoxicoses can be preventedby the use of vZ-bromo-S-nitrothia- Zole in less than fungicidalconcentrations. anism of activity is unknown; however, it is believedthat small amounts of the Z-bromo-S-nitrothiazole interfere with themetabolism of the fungi and thereby prevent the elaboration of toxicby-products. While there may be a proliferation of the fungi in thecereal, a subsequent ingestion of the infected cereal with less thanfungicidal concentrations of .Z-bromo-S-nitrothiazole does not producemanifestations of mycotoxicoses.

Therefore, the invention relates to a cereal composition, containing acereal which when infected with fungi is normally capable of producingtoxic levels of metabolic by-products in conditions of relatively highhumidity, and a concentration of 2-bromo-5-nitrothiazole, which iswithin the range of 10 to 500 ppm.

For the reasons outlined above, a concentration of 10 to 400 ppm. inpoultry feed, for example, does not necessarily suppress profuse fungalproliferation in inoculated feed; however, an ingestion of this feed bychicks does not result in any manifestations of the poultry hemorrhagicsyndrome. Therefore, although there may be profuse fungal proliferationin the inoculated feed, there is sufficient suppression of the poultryhemorrhagic syndrome.

Although the invention is not limited to a particular group of fungi,the following have been observed as being capable of producingmycotoxicoses. Typical of such fungi are: Aspcrgillus clawltus,Aspergiltus flavus, Aspargillus c/zevulieri, Aspergillus fumigulur,Penicillin/71 rubrum, Pcnicillium purpurogenum, other strains ofPenicillium and Alternaria species. Certain Fusarium and Mucorales arealso sometimes found. Undoubtedly there exist other toxic fungi whichhave not as yet been isolated and identified. Mycotoxicoses have beensuppressed by the use of a 2-bromo-5-nitrothiazole in concentrations ofTrue mech 3 to 500 p.p.m., in cereals which have been inoculated withsuspensions of the aforementioned fungi.

The following examples are provided for illustrative purposes and mayinclude particular features of the invention. However, the examplesshould not be construed as limiting the invention, many variations ofwhich are possible without departing from the spirit or scope thereof.

EXAMPLE l.--PREPARATI-ON OF ANIMAL FEED FOR HEMORRHAGIC SYNDROME STUDYEight kilograms of non-sterile broiler mash were weighed and themoisture content of the feed determined by means of a Delmhorst MoistureDetector. To'the sample were added 40 ml. of an aqueous suspension of amixture of toxic fungi (Alternaria 17., Aspergillus clavams, Aspergillusfumigatus, Aspergillus flavus, Aspergiilus glaucus, Fenicillium rubrumand two unidentified species of Penicillia) and sufficient tap waterfree of chlorine to bring the total moisture content of the feed toapproximately 21 percent. The fungi had been isolated from feed andlitter obtained from broiler houses in which the poultry hemorrhagicsyndrome was enzootic. The moistened sample was then premixed by hand,rubbed through a 1/: inch mesh stainless steel screen and mixedmechanically for 5 minutes in a Hobart feed mixer.

The sample was then divided into two lots, and to a small portionof oneof the lots 8 small quantity of 2- bromo-S-nitrothiazole was added.After thoroughly premixing the 2-bromo-5nitrothiazole with a spatula,more feed was added from the lot from which the small portion was taken,and premixed by hand until the whole lot was premixed, and this wascontinued until the 2-bromo-5- nitrothiazole content was 300 p.p.m.,based on the weight of the moist feed. The lot containing the Z-bromo-S-nitrothiazole (Lot 2) was then further mixed mechanically for 5 minutesin the Hobart feed mixer. Lot 1, which contained noZ-bromo-S-nitrothiazole, served as a control.

Both sample lots were then transferred to round fiber board containers,equipped with a gauze-covered opening in the lid for ventilation, andincubated at ordinary room temperatures. The sample lots were observedperiodically for gross and microscopic proliferation of fungi.

After 14 days, a very heavy fungal growth and formation of fruitingbodies was observed in Lot 1. After 18 days, there was a heavier fungalgrowth observed and further formation of fruiting bodies irrLot l, andno growth was observed in Lot 2. On the 25th day both,

sample lots were removed and dried for 48 hours in incubators adjustedto approximately 45 C.

EXAMPLE 2.DETERMINATION OF THE EFFI- CACY of Z-BROMO S-NITROTHIAZOLE INTHE PREVENTION OF MYCOTOXICOSES During the early stages of the poultryhemorrhagic syndrome, the affected birds show signs of depression,diarrhea which is frequently bloody, paleness of combs and Wattles,anorexia and varying morbidity and mortality. Autopsy findings in suchbirds reveal hemorrhages and congestion in the musculature of the legs,thighs and breast, and in many other tissues, as well as erosion of themucosa of the proventriculus and gizzard. The other tissue changes arecharacteristic of an acute degenerative condition in the liver andkidneys.

Autopsy findings, in birds which have progressed to the advancedsyndrome, will reveal blood pigments in the liver, proteinaceous andhematogenous casts in the distal convoluted and collected tubules of thekidneys, and

paleness, and gelatinization of, and depression in, the

blood-forming elements in the bone marrow.

Two groups of New Hampshire-Red Barred Rock cross, day-old chicks, 20per group, werefed ad libitum, respectively, the following lots of feed:the inoculum-containing lot with 300 p.p.m. of 2-bromo-5-nitrothiazole,hereinafter called Lot 2, and the inoculum-containing lot, without theZ-bromo-S-nitrothiaable, Lot 1.

On the fourth day of feeding, chicks in the group which were beingmaintained on the feed of Lot 1, showed signs of the early symptoms ofthe hemorrhagic syndrome, that is, depression and fetid diarrhea. Thiscondition prevailed until the 17th day when depression was no longerobserved; however, diarrhea in this group was observed on the 21st day.Thereafter, chicks in this group grossly appeared normal.

The second group of chicks which were maintained on the feed of Lot 2,in which -2-bromo-5-nitrothiazole was added, showed none of the earlysymptoms of the hemor rhagic syndrome. The chicks of this group grosslyappearcd normal for the full period ofth'e test.

Additional lots of feed were prepared following the procedure set forthhereinabove. At the end of four weeks, the supply of feed was exhausted.and 10 chicks from each group were sacrificed by decapitation, andexamined grossly at autopsy for manifestations of the syndrome. I

These test results show'that there is suppression of the hemorrhagicsyndrome. This is shown by the autopsy, as well as by gross observationwhich revealed a pronounced suppression of the hemorrhagic syndrome.

Attempting to achieve greater lucidity, as well as speed, in theinterpretation of the results of the autopsy, an arbitrary hemorrhagicsyndrome score was assigned to the twelve organs examined at autopsy. Anormal organ is given the value zero, slight manifestations 1, moderatemanifestations 2, pronounced manifestations 3. The score is then totaledfor each of the carcasses examined, and a mean score is determined.bydividing the total score of all the carcasses, examined by the numberof carcasses. The results of the autopsy of the two groups of chicks areset forth in the following table:

EXAMPLE 3.COMPARATIVE. STUDIES OF THE EFFICACY OF LESS THAN FUNGICIDALCON- CENTRATIONS OF 2 BROMO 5 -'NITROTHIA- ZOLE AND FUNGICIDALCONCENTRATIONS OF .Z-BROMO-S-NITROTHIAZOLE ON THE SUP- PRESSION OF THEHEMORRHAGIC SYN- DROME A Nine lots of non-sterile broiler mashcontaining no antibiotic of coccidiostat were inoculated with a mixtureof toxic fungi A lterlmria sp., Aspergillus clnvatus, Aspergillus,fumigatus, Aspcrgillus flavus, Aspergillus glaucus, Paecilomycesvarioti, Penicillium purpurogenum, Penticillium rubrum, and threeunidentified species'of, Penicillia), adjusted to 21.8 percent moisture,and containing respectively,'0,'l0, 50, 100, 200,- 250, 300, 400 and 500p.p.m. of 2-brorno-5-nitrothiazole, were. incubated at ordinary roomtemperatures, observed for fungal proliferation over a period of 13 to14 days, then dried and fed to day-old chicks, 10 animals per group. Thechicks, which were maintained on batteries, had free access to water andthe prepared feeds, and were observed daily for gross manifestation oftoxicity and weighcd periodically until the supply of feed becameexhausted in any one group. After 4 weeks the feed supply in one groupbecame exhausted. At'this time all chicks in each group were sacrificedby decapitation and examined at aaoasea no growth 3. slight growth 4,profuse growth As indicated in the following table, the lots 3containing from 0 to 100 ppm. of Z-bromo-S-nitrothiazole showed profusefungal proliferation after 14 d. Slight growth was observed in the lotscontaining 20%.. ppm. of Z-bromo-S-nitrothiazole after 14 days.

No fungal growth was observed in the samples containing 400 and 500 ppm.of Z-bromo-S-nitrothiazole.

Table 2 Growth on Days Indicated, Days Concentration Compound (p.p.m.)

Mani c 0000 a: oOHb-H- coca 0o ci-kybph H 0009 H mamas,

OCHH

It can be conclusively stated, as a result of autopsy findings, thatalthough the 2-brom0-5-nitrothiazole did not suppress fungalproliferation in the lots containing 10-400 ppm. of the compound, thechicks that consumed these lots showed a pronounced suppression of thehemorrhagic syndrome. It has been observed in many experiments thatchicks used as controls and which received s0 called normal feed as itis purchasedon the open market, at autopsy, had an average hemorrhagicscore of 4 to -5. Thus, the mean scores of 5 for 50 p.p.m. level, aswell as those for the 250 ppm. levels and above can be considered toapproximate normal values. It can also be stated that the2-bromo-5-nitrothiazole in the concentrations tested had no apparenteffect on gain in body weight. The results of the autopsy are set forthin the following table, again utilizing the arbitrary hemor- EXAMPLE4.-PRODUCTION OF THE HEMOR- RHAGIC SYNDROME UNDER FIELD CONDI- TIONS Therelationship of the toxic fungal elaboration products to the hemorrhagicsyndrome was confirmed. by an effective production of the hemorrhagicsyndrome under field conditions. The relationship was further validated,by concurrently suppressing the hemorrhagic syndrome under fieldconditions, using the fungicidalconcentration of2-bromo-5-nitrothiazole.

Production of the hemorrhagic syndr0me-.-N0rmal broiler mash having amoisture content of 12.8 percent, containing no antibiotic orcoccidiostat, was inoculated with 0.5 percent of dry inoculum of themixture of toxic fungi described in Example 1. The feed was then fed tochicks maintained on woodshavings litter on a concrete floor in abuilding having a total area of square feet. @ne hundred chicks, 50males and 50 females, had free access to the feed in hoppers and waterin normal watering devices located on the litter. Litter andfeedsubsequently scattered in the litter by the chicks, were examedperiodically by steriomicroscopic means for fungal proliferation, andthe chicks observed for gross manifestation of toxicity.

Nineteen days after initiation of the experiment, slight fungalproliferation was observed in feed scattered by the chicks in litternear water devices, and heavy fungal growth was observed three dayslater. Fungal proliferation gradually became more evident in feedscattered in other parts of the chicken house and was pronounced in feedand litter in all parts of the house by the fourth week.

On the 22nd day of the experiment, slight depression, diarrhea, andpaling of combs and Wattles were observed in some chicks and became morepronounced in approximately 25 percent of the flock at 4 weeks. Deathbegan to occur in chicks at 4 weeks and mortality was 10 percent at 5V2weeks.

Gross autopsy findings were typical of those observed in field cases ofpoultry hemorrhagic syndrome, namely, superficial and deep hemorrhagesand congestion in the musculature of the legs, thighs, and breasts;hemorrhages and congestion in the lungs, liver, spleen, kidneys, smallintestine and caeca, and pronounced erosion in the proventriculus andgizzard. At 7 /2 weeks, when the experiment was concluded, moderatedepression and fetid diarrhea were observed in practically 100 percentof surviving chicks. Five males and five females sacrificed bydecapitation showed gross autopsy findings of severe manifestations ofpoultry hemorrhagic syndrome having an average hemorrhagic syndromescore of 30. i

It was also determined that the hemorrhagic syndrome exists in twoforms, namely the acute and chronic. The acute form appears afteringestion of large quantities of a highly toxic substrate, and ischaracterized for the most part by a hemorrhagic diathesis andploethora, and degenerative changes in the liver and kidneys. When smallamounts of toxic feed are administered over a long period of timeanimals develop various stages of chronic toxicoses. The mostoutstanding symptom of the final stage of the chronic syndrome ispronounced depression of the haemophoeitic system.

Suppression of the hemorrhagic .s'yndr0mc.To conclusively prove therelationship of the toxic fungal metabolic by-products to thehemorrhagic syndrome, it is be lieved that a suppression of the fungalproliferation in feed and litter will concomitantly result in asuppression of the poultry hemorrhagic syndrome in chicks maintainedunder field conditions which would be substantially the same as thosedescribed in the foregoing production of the hemorrhagic syndrome.

To the inoculated feed were added by rubbing through SO-mesh screen, 500p;p.m. of Z-bromo-S-nitrothiazole, mixed for 15 minutes in a cementmixer and fed to 100 chicks.

Steriomicroscopic examination of litter and feed scattered in litter bythe chicks for 9 weeks did not demonstrate fungal proliferation. Inaddition, during this period the chicks demonstrated no evidence ofpoultry hemorrhagic syndrome. Five males and five female chicks weresacrificed at this time by decapitation. The autopsy findings resultedin showing that the chicks for all practical purposes were normal,having an actual average syndrome score of less than 1.0. This is astriking comparison to the fact, that as indicated in Tables 1 and 2,the chicks that consumed the feed with the inoculum alone demonstratedan average syndrome score of 16.

I claim: capable of producing toxic levels of metabolic by-prodl. Amethod of so treating cereals as to prevent the nets in conditions ofrelatively high humidity containing formation therein, by the action offungi in the presence 21 concentration of 2-bromo-5-nitrothiazole, whichis withof moisture of amounts of metabolic by-products that will in therange of 10-500'p.p.m. be toxic to animals ingesting the same, whichcomprises 5 4. A composition according to claim 3 in which theincorporating into said cereal 10-500 pip.m. of Z-bromoconcentration of2-bromo-5-nitrothiazole is within the 5-n 1 range of 30-300 p.p.m.

Z. A method according to claim 1 in which the concen- I iration of2-bromo-5-nitrothiazole is'within the range oi No references cited.

30-300 ptpJn. m

3.- A cereal, which when infected with fungi is normally LOUIS MONACELL,Examiner-

1. A METHOD OF SO TREATING CEREALS AS TO PREVENT THE FORMATION THEREIN, BY THE ACTION OF FUNGI IN THE PRESENCE OF MOISTURE OF AMOUNTS OF METABOLIC BY-PRODUCTS THAT WILL BE TOXIC TO ANIMALS INGESTING THE SAME, WHICH COMPRISES INCORPORATING INTO SAID CEREAL 10-500 P.P.M. OF 2-BROMO5-NITROTHIAZOLE.
 3. A CEREAL, WHICH WHEN INFECTED WITH FUNGI IS NORMALLY CAPABLE OF PRODUCING TOXIC LEVELS OF METABOLIC BY-PRODUCTS IN CONDITIONS OF RELATIVELY HIGH HUMIDITY CONTAINING A CONCENTRATION OF 2-BROMO-5-NITROTHIAZOLE, WHICH IS WITHIN THE RANGE OF 10-500 P.P.M. 